Structure-function studies of the cellular retinaldehyde-binding protein (CRALBP) are proposed that focus upon functional domain analysis and development of protein preparations suitable for three dimensional structural analysis. The hypothesis of the proposal is that CRALBP plays a fundamental role in the metabolism of vitamin A in the visual cycle. CRALBP is a substrate carrier protein found only in visual tissue that carries 11-cis-retinol and 11-cis-retinaldehyde, retinoids that are only known to function in vision. The protein appears to play a regulatory role, influencing whether 11-cis-retinol is stored as retinyl ester in the retinal pigment epithelium (RPE) or oxidized to 11-cis-retinaldehyde and exported for visual pigment regeneration in photoreceptor cells. CRALBP stimulates oxidation and retards esterification in vitro, suggesting that the protein may help control retinoid flow at a key metabolic branch point in the visual cycle. A defect in the synthesis, regulation or structure of CRALBP could compromise vitamin A metabolism, resulting in impaired vision. Functional domain analysis will utilize human recombinant CRALBP and apply amide hydrogen/deuterium exchange and mass spectrometry for topological analysis to facilitate identification of buried domains associated with retinoid-binding and exposed regions involved with protein-protein interactions. Photoaffinity-labeling of rCRALBP will be performed to identify the retinoid-binding pocket and the crosslinked sites determined by Edman degradation and electrospray mass spectrometry. Targets for site-directed mutagenesis will be based upon results from these approaches and changes in specific amino acids used to define residues critical for retinoid-binding and interaction with 11-cis-retinol dehydrogenase. Limited alanine scanning mutagenesis will also focus around two residues in a random rCRALBP mutant that has lost the ability to bind retinoid. A procedure will be developed for crystallizing human rCRALBP for the long term goal of 3D structural determination by crystallography. Limited proteolysis will be used to identify the smallest fragment capable of binding retinoid. The retinoid-binding fragment will also be produced as a recombinant protein for solution structural analysis by NMR. The substrate carrier efficacy of proteolysis fragments and mutants will be assayed using bovine RPE microsomes as the source of 11-cis-retinol dehydrogenase. For comparative purposes, native bovine GRALBP will be used as a structure-function reference. The unifying hypothesis of the research is that by establishing a molecular basis for understanding the normal structure, function and regulation of CRALBP, questions concerning visual disorders with which the protein may be associated can be answered in specific terms.